Targeted analysis of protein citrullination using chemical modification and tandem mass spectrometry

Abstract

Protein citrullination originates from enzymatic deimination of polypeptide-bound arginine and is involved in various biological processes during health and disease. However, tools required for a detailed and targeted proteomic analysis of citrullinated proteins in situ, including their citrullination sites, are limited. A widely used technique for detection of citrullinated proteins relies on antibody staining after specific derivatization of citrulline residues by 2,3-butanedione and antipyrine. We have recently reported on the details of this reaction. Here, we show that this chemical modification can be utilized to specifically detect and identify citrullinated peptides and their citrullination sites by liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis. Using model compounds, we demonstrate that in collision-induced dissociation (CID) a specific, modification-derived fragment ion appears as the dominating signal at m/z 201.1 in the MS/MS spectra. When applying electron transfer dissociation (ETD), however, the chemical modification of citrulline remained intact and extensive sequence coverage allowed identification of peptides and their citrullination sites. Therefore, LC/MS/MS analysis with alternating CID and ETD has been performed, using CID for specific, signature ion-based detection of derivatized citrullinated peptides and ETD for sequence determination. The usefulness of this targeted analysis was demonstrated by identifying citrullination sites in myelin basic protein deiminated in vitro. Combining antibody-based enrichment of chemically modified citrulline-containing peptides with specific mass spectrometric detection will increase the potential of such a targeted analysis of protein citrullination in the future.

Methods for the Detection of Peptidylarginine Deiminase (PAD) Activity and Protein Citrullination

“…Phenylglyoxal-rhodamine Probe-Another method that was recently described to be applicable for the detection of citrullinated proteins is based on the chemical reaction of the ureido group of citrulline with phenylglyoxal (36,37). Phenylglyoxal has been reported to react specifically with arginine residues under neutral or basic conditions and with citrulline residues under highly acidic conditions.…”

“…However, this approach did not allow the identification of the site of citrullination. They optimized the procedure further and subjected the chemically modified citrullinated peptides to LC-MS/MS with alternating CID and ETD (36). In this way, citrullinated peptides were detected in the CID spectrum, which showed a dominant peak with an m/z value of 201.1, corresponding to a specific modification-derived fragment ion, and the exact site of citrullination could be determined from the ETD spectrum.…”

Proteomics in chromatin biology and epigenetics: Elucidation of post-translational modifications of histone proteins by mass spectrometry

“…This modification is catalyzed by peptidylarginine deiminases (PADs) [59], and is often related to gene silencing, since it antagonizes arginine methylation [60]. From an analytical perspective chemical tags such as 18 O, 2,3-butanedione, or antipyrine are useful to convert protein citrullination sites into derivatives for efficient and robust MS detection [61,62], since the mass increment of deimination (+0.984 Da) is rather small and may be obscured by isotope splitting of the peptide signals in MS and MS/MS.…”

Safe exposure level for diacetyl

“…12 Diacetyl is used to react with protein-bound arginine to produce protein-bound citrulline. 13,14 Researchers have treated citrullinated residues with diacetyl and antipyrine to create citrullinated proteins which react with anti-cyclic citrulline protein antibodies (ACPAs). 13,15 Recently several authors have suggested that ACPA production accompanies or contributes to…”